Miguel Cantamutto

Gene flow between cultivated sunflower and Helianthus petiolaris (Asteraceae)

Helianthus petiolaris (Asteraceae) native to North America has naturalized in Argentina. The extensive overlapping with sunflower crop regions, the coincidence of life cycles and the common pollinators facilitate interspecific crosses with sunflower, H. annuus var. macrocarpus. To estimate the occurrence of crop-to-wild and wild-to-crop gene flow, off-type plants in progenies of pure stands of both species flowering in coincidence and the presence of crop alleles in H. petiolaris populations were investigated in 26 wild populations and nine crop fields. Morphological traits and RAPD markers were used to attempt hybrid characterization. Off-type individuals were found in frequencies of 0.5 and 0.3% among crop progenies and wild populations, respectively. Off-type plants showed intermediate values for metric morphological traits and low fertility traits. Some off-type plants proved to carry crop alleles based on molecular analysis. The average frequency of cultivar-marker introgression across the wild populations was very low (0.02). Although observed hybridization rates seem to be low, the extension of crop-wild overlapping in Argentina make hybrid formation a noticeable process. Therefore, the new sunflower varieties and eventually GM varieties would transfer their traits through pollen flow and they would persist in H. petiolaris populations.

Wild helianthus species and wild-sunflower hybridization in Argentina

SUMMARY Two wild Helianthus species native to North America have been naturalized in Argentina, H. annuus ssp. annuus and H. petiolaris. They grow as adventitious overlapping about 50% of the crop area. Hybridization and introgression between these wild species and sunflower have important biological and practical consequences, the former including homoploid hybrid species formation, and the latter including a possibility of transgene spreading from genetically modified (GM) sunflower cultivars to wild or weedy populations. Wild populations were screened for isozyme and morphological variation. Intermediate plants were found in several locations and subjected to progeny tests. Variability among progeny of each one was compared with the variability in wild accessions of both species. Segregation for phenotypic traits, intermediate phenology and low fertility levels were found in most progenies, accounting for the hybrid origin of their maternal plants. Attempts to quantify gene flow included screening of progenies from H. petiolaris populations growing near sunflower crops. Hybrid plants were recognized by morphological traits and reduced fertility. Overall hybridization was about 1%. Hybrid progeny on wild H. annuus plants were identified by a crop isozyme marker. A mean frequency of 7% hybridization was found. These results confirm that gene flow occurs among crop and wild Helianthus species, and it concerns crop management and environmental impact if release of GM sunflower cultivars is to be authorized in Argentina. Resumen Dos especies de Helianthus nativas de América del Norte se han naturalizado en Argentina, H. annuus ssp. annuus y H. petiolaris y crecen coincidiendo en un 50% con el área cultivada. La hibridación e introgresión entre estas especies silvestres y el girasol cultivado tienen importancia biológica y consecuencias prácticas, en la formación de híbridos homoploides y en la posibilidad de escape de transgenes desde cultivos de girasol genéticamente modificados (GM) a las especies silvestres o malezas. Se evaluó la variación morfológica e isoenzimática de las poblaciones silvestres. En diferentes localidades se encontraron plantas intermedias y algunas se sometieron a pruebas de progenie. La variabilidad encontrada dentro de las progenies fue comparada con la variabilidad presente en poblaciones silvestres de ambas especies. La mayor parte de las progenies mostró segregación para rasgos fenotípicos, fenología intermedia y niveles bajos de fertilidad, demostrando el origen híbrido de las plantas madres. A fin de cuantificar el flujo génico, se estudiaron progenies de poblaciones de H. petiolaris que crecían cercanas a cultivos de girasol. Las plantas híbridas fueron reconocidas por rasgos morfológicos y reducida fertilidad. El total de híbridos encontrados fue de 1%. La progenie híbrida en plantas de H. annuus silvestre fue identificada por un marcador isoenzimático. Se encontró una frecuencia promedio de hibridación del 7%. Estos resultados confirman que ocurre el flujo génico entre el cultivo y las especies silvestres de Helianthus lo cual tiene importancia en el manejo del cultivo y el impacto ambiental, si se aprobara la liberación de cultivos de girasol genéticamente modificados en Argentina. RÉSUMÉ Deux espèces sauvages du genre Helianthus indigènes d’Amérique du Nord ont été naturalisées en Argentine, H. annuus ssp. annuus et H. petiolaris, et elles se répandent comme adventices en recouvrant environ 50% de l’aire cultivée. L’hybridation et l’introgression entre ces deux espèces sauvages et le tournesol cultivé ont une importance biologique et des conséquences pratiques dans la formation d’hybrides homoploïdes et dans la possibilité de dissémination de transgènes depuis des cultures de tournesol génétiquement modifiés (GM) vers les espèces sauvages ou envahissantes. Les populations sauvages ont été évaluées dans sa variabilité morphologique et des isozymes. Des plantes intermédiaires ont été trouvées dans plusieurs localités et on été soumises à des tests de descendance. La variabilité trouvée dans les descendances a été comparée avec la variabilité dans les populations sauvages des deux espèces. La ségrégation pour des caractères phénotypiques, la phénologie intermédiaire et des bas niveaux de fertilité ont été trouvés dans la plupart des descendances, en accord avec l’origine hybride des plantes mères. Avec le but de quantifier le flux de gènes ont a inclus l’étude des descendances de H. petiolaris qui se développaient proches des cultures de tournesol. Les plantes hybrides ont été reconnues par des caractères morphologiques et une fertilité réduite. La totalité d’hybrides trouvés a été de 1%. La descendance hybride dans les plantes H. annuus sauvages a été identifiée par la présence d’un isozyme spécifique du tournesol cultivé. On a trouvé une fréquence moyenne d’hybridation de 7%. Ces résultats confirment que le flux de gènes se produit entre les espèces cultivée et sauvages de Helianthus, ce qui est important pour la gestion des cultures et l’impact environnemental si on admet le lancement des cultivars de tournesol GM en Argentine.

Ecological characterization of wild Helianthus annuus and Helianthus petiolaris germplasm in Argentina.

Fil: Poverene, Maria Monica. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Bahia Blanca. Centro de Recursos Naturales Renovables de la Zona Semiarida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiarida; Argentina

Development of a thermal-time model for combinational dormancy release of hairy vetch (Vicia villosa ssp. villosa)

Abstract. Seed dormancy could be a factor related to natural reseeding of hairy vetch (Vicia villosa ssp. villosa Roth.), a winter annual species cultivated for seed, pasture, hay, green manure and cover crop. The presence of combinational dormancy (physical dormancy + physiological dormancy, PY + PD) in hairy vetch was explored by a model using laboratory and field measures. At the stage of natural dispersal, dry seeds of hairy vetch were stored under laboratory conditions at 5, 10, 20 and 30°C (±2°C) or buried at 5 cm depth in an experimental field. Germination at 5, 8, 10, 15, 20 and 25°C was assessed at regular intervals up to 295 days after harvest. Following the hypothesis of the existence of a combinational dormancy mechanism, model development was based on the estimation of: (i) the fraction of non-PY seed as a function of after-ripening thermal-time accumulation, and (ii) seed population thermal parameters associated with a given level of PD. The developed model adequately described the after-ripening thermal-time requirements for PY + PD release of V. villosa. Based on model predictions, under a semi-arid thermal regime, >45% of vetch seeds shed during the summer season would be able to germinate during early autumn. Thus, the seed-bank size threshold at the end of the first growing season should be >65 seeds m–2 in order to reach a minimum stand of 30 plants m–2 necessary for a productive pasture.

Limited occurrence of resistant radish (Raphanus sativus) to AHAS-inhibiting herbicides in Argentina

Radish has developed feral and weedy biotypes, which is a concern for agriculture around the world. In Argentina, it is one of the most widespread and troublesome crop weeds. In Brazil, this species has developed herbicide-resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides. The objective of this study was to record the presence of herbicide-resistant weedy radish plants in Argentina. In spring 2008, we found a small population of radish at the end of the flowering stage in an imidazolinone-tolerant canola field treated with imazethapyr. Screening and dose-response tests were conducted to two successive generations. They proved the biotype resistant status, and showed extensive survival (between 50 and 80% of control) to the application of a double dose of four AHAS‑inhibiting herbicides from two different chemical families (imidazolinones and sulfonylureas). Dose-response assays exhibited very high resistance for imazethapyr (LD50 = 2452.5 g a.i. ha-1, GR50 = 2926.9 g a.i. ha-1) and intermediate for metsulfuron (LD50 = 3.0 g a.i. ha-1, GR50 = 43.2 g a.i. ha-1). The acquisition of cross-resistance to different herbicide families would confer an adaptive and invasive advantage in agricultural environments to this biotype. Due to the herbicide rotation conducted in the field, the dispersion of this biotype was restricted. This is the first report of resistance in weedy radish in Argentina.

Broad resistance to acetohydroxyacid-synthase-inhibiting herbicides in feral radish (Raphanus sativus L.) populations from Argentina.

BACKGROUND Soon after the commercial release of sunflower cultivars resistant to imidazolinone herbicides, several uncontrolled feral radish (Raphanus sativus L.) populations were found in south-eastern Buenos Aires, Argentina. These populations were studied in field, glasshouse and laboratory experiments aiming to characterise their resistance profile and to develop management tools. RESULTS Three feral radish accessions were highly resistant to ten active ingredients of five families of acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Sequence analysis of the AHAS gene detected a Trp574Leu mutation in all resistant accessions. One accession with an intermediate level of resistance was heterozygous for this mutation, probably owing to gene exchange with a susceptible subpopulation located in the field margin. Herbicide-resistant and herbicide-susceptible radish could be controlled in sunflower by alternative herbicides. CONCLUSION This is the first report of feral radish with resistance to herbicides belonging to all the AHAS-inhibiting herbicide families, conferred by Trp574Leu mutation in the AHAS gene. An appropriate herbicide rotation with alternative herbicides such as fluorochloridone or aclonifen and an increase in the diversity of cropping systems are important for minimising the prevalence of these biotypes.

AHAS Trp574Leu substitution in Raphanus sativus L.: screening, enzyme activity and fitness cost: Fitness cost in AHAS-resistant Raphanus sativus

BACKGROUND Feral radish (Raphanus sativus L.) is a problematic weed that has become resistant to acetohydroxyacid synthase (AHAS) inhibitor herbicides due to the Trp574Leu mutation. An AHAS gene mutation that causes herbicide resistance may have negative pleiotropic effects on plant fitness. This study reports the effects of the Trp574Leu mutation on AHAS activity and reproductive traits of R. sativus. RESULTS Eight of 17 feral radish accessions presented individuals resistant to metsulfuron-methyl at 0.5% to >90.0% and all the resistant individuals analyzed showed the Trp574Leu mutation. Without herbicide selection, the AHAS activity was 3.2-fold higher in the susceptible accession than in the resistant one. The resistant accession was >9000-fold more resistant to metsulfuron-methyl and imazethapyr than the susceptible accession. Under low intraspecific competition during two growing seasons, AHAS-resistant feral radish accessions showed 22-38% and 21-47% lower seed numbers and yield per plant than the susceptible accession. CONCLUSION This is the first report of fitness cost associated with the AHAS Trp574Leu mutation in R. sativus populations. This fitness cost could reduce frequency of the resistant allele without herbicide selection.

Effect of water availability and seed source on physical dormancy break of Vicia villosa ssp. villosa

Physical dormancy (PY) plays a crucial role in the control of the reseeding process of Vicia villosa Roth, a winter annual species cultivated for pasture and hay, naturalized in several semi-arid temperate agroecosystems. As PY is considered a seed trait modulated by natural selection, populations from different origins are expected to show different responses to environmental regulatory factors. The present study aimed to determine the effect of: (1) water availability on PY-break dynamics of a naturalized population from Argentina (ASC) under both laboratory and field conditions; (2) the seed source on initial PY and dormancy release rate (wet storage at 20°C) of ASC compared to 45 other populations of V. villosa , including wild, naturalized, landraces and cultivars. Water availability increased PY loss rate under both storage and field conditions. ASC PY-break dynamics was adequately described by a Gompertz model with a lower thermal-time requirement estimated for dormancy break under fluctuating soil water conditions compared to seeds buried inside impermeable bags. During the field burial experiment, a considerable proportion of seeds (~70%) became water permeable during the summer season after dispersal, and retained low levels of residual PY for soil seed bank replenishment. Improved populations (i.e. breeding cultivars) showed the lowest percentages of initial PY compared to landraces, naturalized and wild populations. Naturalized populations of Argentina showed similar initial PY compared to landraces, although PY release rate was lower in the former and might be attributed to local environmental selection. Wild types showed the lowest PY release rates.

Disease tolerance in Helianthus petiolaris: a genetic resource for sunflower breeding.

Abstract Argentina is a major sunflower producer in the world, with crop acreage of 2−2.7 million ha in the last four years. Sunflower crop yield is often influenced by sanitary constraints, mainly fungal pathogens. Helianthus petiolaris is a wild species native to North America established in central Argentina displays a high tolerance to a number of fungal diseases and insects. Controlled crosses of this species with sunflower demonstrated that H. petiolaris constitutes a valuable genetic variability source for sunflower breeding to improve tolerance to rust (Puccinia helianthi), white rust (Albugo tragopogonis), verticillium wilt (Verticillium dahliae), powdery mildew (Erisiphe sp.) and the sunflower moth (Rachiplusia nu). This places H. petiolaris in an outstanding position as a genetic resource since different important traits could be transferred to the crop through interspecific hybridization.

Evaluation of Argentine wild sunflower biotypes for drought stress during reproductive stage

SUMMARY As in several regions where sunflower production has moved to areas with limiting water availability, the search for water-stress tolerant genotypes has been intensified. Helianthus annuus ssp. annuus L. constitutes a potential genetic resource because it has naturalized in the semi-arid zone of central Argentina. The assessment of these genetic materials for water deficit tolerance is of interest because they may represent a source of genes for drought tolerance, useful to sunflower breeding. Drought resistant genotypes should be achieved using easily identified phenotypic traits. Parameters like leaf area are widely used to characterize the performance under stress. Leaf temperature is an easily measured physiological parameter that allows an indirect estimate of plant transpiration and is well correlated with water availability. Relative water content indicates the ability to retain water from the soil and expresses plant osmotic adjustment ability. Specific leaf area is a morphological parameter related to leaf thickness. The objectives of this study were to evaluate the drought tolerance of Argentine wild sunflower biotypes and identify morphological and physiological traits expressing differences between stressed biotypes. Wild biotypes were evaluated during three years in the experimental field of the Agronomy Department, Universidad Nacional del Sur, Argentina. Groups of 10-15 plants of each biotype were evaluated under two water conditions, drought (deficit supply) with drip irrigation to cover a half of the potential evapotranspiration during flowering, or with optimal water supply. Soil surface was covered with black polyethylene to exclude rainwater. Wild sunflower comprised five biotypes collected from different habitats in the semiarid region of Argentina. Crosses between the wild biotypes and inbred lines were also included every year. Inbred lines and a commercial hybrid (DK4000) were used as controls. Recorded traits were: plant height, stem diameter, petiole length, leaf area, leaf number, head number and reproductive surface. Leaf parameters were: relative water content (RWC), specific leaf area (SLA), canopy temperature (CT), and chlorophyll content (SPAD). A susceptibility index (SI) was obtained to compare the performance under water stress with that obtained in optimal conditions. Biotype evaluation and parameter characterization were performed separately for each year because water stress levels were different. Wild sunflower responses to water stress were different for all parameters among biotypes, except for plant height and petiole length. Wild biotypes had better RWC and lower SLA than cultivated biotypes. Under water stress wild biotypes showed higher values and greater range of RWC and SPAD than cultivated sunflower. Susceptibility index showed that leaf area of wild sunflower biotypes had lower stress susceptibility than cultivated sunflowers. Nevertheless, wild biotypes showed increased susceptibility to the remaining plant morpho-physiological parameters. RWC and CT had a significant relationship in wild sunflower biotypes under water stress. Drought tolerant type identification was complex because of the complex responses among parameters. Wild biotypes might have a physiological mechanism which allows higher RCA than cultivated sunflower under drought stress. The lower SLA under water deficit could be attributed to a greater leaf thickness and could be related with RCA. Lower leaf area reduction under stress in wild sunflower is an interesting trait that might be used to improve cultivated sunflower. As the RWC is related with CT under stress in wild biotypes, this trait evaluation allows the fast examination of a high number of plants. The assessment of Argentina wild sunflower biotypes for traits associated with drought tolerance has not yet been done. Their identification could increase sunflower crop yield under drought in semiarid regions.